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E Salih

Publications and source records attributed to E Salih.

At least 37 records · Page 2Linked to original sources
PubMed1995

Characterization of the major non-collagenous proteins of chicken bone: identification of a novel 60 kDa non-collagenous phosphoprotein.

Chicken bone powder, sequentially extracted with 4M guanidine HCl (NG), 0.3M HCl (H), 0.5 NaCl at neutral pH (NS), and 4M guanidine HCl pH 7.4 (G), preferentially solubilized osteocalcin (OC), osteopontin (OPN), alpha 2 HS-glycoprotein, bone sialoprotein (BSP), and osteonectin (ON). These studies provide the first amino acid sequences for a non-mammalian form of ON and both N-terminal and internal amino acid sequences obtained for ON demonstrated that the avian form of this protein is more than 80% conserved for all the peptides sequenced in this study. A novel approximately 60 kDa protein was identified with a unique N-terminal amino acid sequence and several internal amino acid sequences. Amino acid composition of this protein indicates that it is similar to other acidic glycosylated phosphoprotein of bone, and it is phosphorylated principally on serine residues, although a small amount of phosphorylated threonine residues were also detected. Solubility characteristics of this protein differed from those of ON, OPN and Decorin, and, it was not recognized by polyclonal antibodies to ON, BSP, OPN or by a monoclonal antibody to Decorin. This protein was not present in chicken serum and no comparable sequences were found in the GenBank or EMBL protein sequence database. These studies provide the first sequence for a non-mammalian form of ON and identify a novel acidic bone protein.

Amino Acid Sequence↗
PubMed1994

Active-site peptides of acetylcholinesterase of Electrophorus electricus: labelling of His-440 by 1-bromo-[2-14C]pinacolone and Ser-200 by tritiated diisopropyl fluorophosphate.

To characterize the structure of the active site of acetylcholinesterase (AChE) from the electric organ of E. electricus, we identified sites of incorporation of two active-site affinity labels, [3H]diisopropyl fluorophosphate ([3H]DFP), and 1-bromo-2-[14C]pinacolone ([14C]BrPin). AChE was isolated, purified, inactivated and digested with trypsin, and peptides containing 3H or 14C were purified by reverse-phase HPLC and characterized by N-terminal sequence analysis. [3H]DFP, labelling Ser-200, was found in a single peptide, QVTIFGESAGAASVGMHLLSPDSR, 83% identical with the sequence from Thr-193 to Arg-216 deduced for AChE of T. californica, with Gln, Ala, Leu, and Asp in place of Thr-193, Gly-203, Ile-210 and Gly-214, respectively, and 87% identical with that from bovine and human brain AChEs. Inactivation by [14C]BrPin led to two radioactive peptides. One, ASNLVWPEWMGVIHGYEIEFVFGLPLEK, was 96% identical with that extending from Ala-427 to Lys-454 of T. californica. Release of 14C in cycle 14 established reaction of [14C]BrPin with active-site His-440, protected by 5-trimethylammonio-2-pentanone (TAP). The other peptide, LLXVTENIDDAER, 77% homologous with that of T. californica extending from Leu-531 to Arg-543, had label associated with the third cycle, not protected by TAP, corresponding to Asn-533. The slow inactivation of eel AChE by reaction of [14C]BrPin at His-440 contrasts with that of AChE from T. nobiliana, where it reacts rapidly with a free cysteine, Cys-231, not present in eel AChE. For both AChEs, inactivation by BrPin prevents subsequent reaction with [3H]DFP, and prior inactivation by DFP does not prevent reactions with [14C]BrPin.

Acetylcholinesterase↗
PubMed1993

Labeling of cysteine 231 in acetylcholinesterase from Torpedo nobiliana by the active-site directed reagent, 1-bromo-2-[14C] pinacolone. Effects of 2,2'-dipyridyl disulfide and other sulfhydryl reagents.

Acetylcholinesterase (AcChE, EC 3.1.1.7) was isolated from the electric organ of T. nobiliana and treated with the active-site-directed alkylating agent 1-bromo-2-[14C]pinacolone ([14C]BrPin), or with BrPin, which acts initially as a competitive inhibitor, Ki = 0.18 mM, and then inactivates the enzyme, k2 = 1.8 x 10(-4) s-1. AcChE aliquots were digested with trypsin and fractionated by reversed phase high performance liquid chromatography. Inactivation caused a decrease in one absorption peak and an increase in another, identified as the peptide beginning at Ala-222 and extending to Arg-242. 5-Trimethylammonio-2-pentanone, a competitive inhibitor, isosteric with acetylcholine, retarded the inactivation and decreased the quantity of labeled peptide. On sequencing, the 14C label was found associated with Cys-231. This was confirmed by comparison with synthesized S-pinacolonylcysteine, by study of effects of blocking the sequencing by o-phthalaldehyde, and by inactivation by 2,2'-dipyridyl disulfide (2-PDS), a thiol-specific reagent that acts initially as a competitive inhibitor, Ki = 0.042 mM, and then inactivates the enzyme, k2 = 5.0 x 10(-4) s-1. This is retarded by 5-trimethylammonio-2-pentanone, and prior inactivation by 2-PDS prevents subsequent reaction of [14C]BrPin in the active site. BrPin inactivates AcChEs from Electrophorus electricus and from human erythrocyte, but 2-PDS does not. Neither reagent inactivates butyrylcholinesterases from human and horse serum.

2,2'-Dipyridyl↗
PubMed1992

Catalysis by acetylcholinesterase in two-hydronic-reactive states. Integrity of deuterium oxide effects and hydron inventories.

Low 2H2O effects (1.0-1.5) for the parameter k(cat.)/Km in the hydrolysis of various substrates by acetylcholinesterase (AcChE) is due to normal 2H2O effects (1.8-2.8) for the parameter k(cat.) and 2H2O effects of 1.0-2.5 for the parameter Km. The analyses and interpretations of 2H2O effects in the literature utilizing the parameter k(cat.)/Km, which led to the proposal of 'isotope insensitivity' of the catalytic steps and the hypothesis of a rate-limiting substrate-induced-fit conformational change, are incorrect. Since k(cat.) is the only parameter that can represent the hydron-transfer step solely, the 2H2O effect can most appropriately be evaluated by using this parameter. Calculations and comparison of acylation (k+2) and deacylation (k+3) rate constants show that acylation is rate-determining for most substrates and the improved binding -0.84 to -2.09 kJ/mol (-0.2 to -0.5 kcal/mol) in 2H2O obscures the normal 2H2O effect on k(cat.) when the ratio k(cat.)/Km is utilized. Consistent with this, measurements of the inhibition constant (KI(com.)) for a reversible inhibitor, phenyltrimethylammonium, lead to KI(com.)H2O = 39 +/- 3 microM and KI(com.)2H2O = 24.5 +/- 3.5 microM, an 2H2O effect of 1.59 +/- 0.26. pH-dependence of k(cat.) in 2H2O is subject to variability of the pK(app.) values, as evaluated in terms of the two-hydronic-reactive states (EH and EH2) of AcChE, and is due to an uneven decrease in 2H2O of the kinetic parameters k'cat. for the EH2 state relative to k(cat.) for the EH state, thus leading to variable shifts in pK(app.) values of between 0.5 and 1.2 pH units for this parameter. The observed pH-independent limiting rate constants for k(cat.)/Km(app.) are made to vary between 0.5 and 1.0 in 2H2O by effects on kinetic parameters for the EH2 state, k'cat./K'm varying between 0.2 and 0.7 relative to the EH state, with k(cat.)/Km varying between 0.4 and 1.0. The effects observed on k(cat.)/Km(app.) are ultimately the result of variable effects of 2H2O on k'cat. and K'm for the EH2 state relative to k(cat.) and Km for the EH state of AcChE. These effects are responsible for the variable shifts and more than 0.5 pH unit of the pK(app.) values in 2H2O for pH-k(cat.)/Km profiles. The upward-bowing hydron inventories for k(cat.)/Km are the result of linear hydron inventories for k(cat.) and downward-bowing on Km and are not due to the rate-limiting substrate-induced fit process as claimed in the literature.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase↗
PubMed1991

Structure-function relationships in the cysteine proteinases actinidin, papain and papaya proteinase omega. Three-dimensional structure of papaya proteinase omega deduced by knowledge-based modelling and active-centre characteristics determined by two-hydronic-state reactivity probe kinetics and kinetics of catalysis.

1. A model of the three-dimensional structure of papaya proteinase omega, the most basic cysteine proteinase component of the latex of papaya (Carica papaya), was built from its amino acid sequence and the two currently known high-resolution crystal structures of the homologous enzymes papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). The method used a knowledge-based approach incorporated in the COMPOSER suite of programs and refinement by using the interactive graphics program FRODO on an Evans and Sutherland PS 390 and by energy minimization using the GROMOS program library. 2. Functional similarities and differences between the three cysteine proteinases revealed by analysis of pH-dependent kinetics of the acylation process of the catalytic act and of the reactions of the enzyme catalytic sites with substrate-derived 2-pyridyl disulphides as two-hydronic-state reactivity probes are reported and discussed in terms of the knowledge-based model. 3. To facilitate analysis of complex pH-dependent kinetic data, a multitasking application program (SKETCHER) for parameter estimation by interactive manipulation of calculated curves and a simple method of writing down pH-dependent kinetic equations for reactions involving any number of reactive hydronic states by using information matrices were developed. 4. Papaya proteinase omega differs from the other two enzymes in the ionization characteristics of the common (Cys)-SH/(His)-Im+H catalytic-site system and of the other acid/base groups that modulate thiol reactivity towards substrate-derived inhibitors and the acylation process of the catalytic act. The most marked difference in the Cys/His system is that the pKa for the loss of the ion-pair state to form -S-/-Im is 8.1-8.3 for papaya proteinase omega, whereas it is 9.5 for both actinidin and papain. Papaya proteinase omega is similar to actinidin in that it lacks the second catalytically influential group with pKa approx. 4 present in papain and possesses a catalytically influential group with pKa 5.5-6.0. 5. Papaya proteinase omega occupies an intermediate position between actinidin and papain in the sensitivity with which hydrophobic interaction in the S2 subsite is transmitted to produce changes in transition-state geometry in the catalytic site, a fact that may be linked with differences in specificity in P2-S2 interaction exhibited by the three enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Two-hydronic-reactive states of acetylcholinesterase, mechanistically relevant acid-base catalyst of pKa 6.5 and a modulatory group of pKa 5.5.

Variation of experimentally observed pKa values in pH-dependent kinetic studies using acetylcholinesterase (AcChE) is rationalized by proposal of two-hydronic-reactive states, EH and EH2, of the free AcChE molecule. Two kinetically influential ionizations with pKa 6.5 for the general acid-base catalyst, possibly the imidazole group of histidine, and a modulatory group with pKa 5.5 residing at the juxtaposal modulatory site, provided fundamental bases for the observed variation in pK(app) values. Appropriate equations applicable to the proposed kinetic model in conjunction with pKa values (pKI 5.5, pKII 6.5) and relative varied values of the pH-independent rate constants, k'cat/K'm and kcat/Km, of the reactive states were used to generate computer simulation error-free pH-rate profiles. A series of theoretical apparently simple sigmoidal pH-rate profiles with characterizing parameters pK(app) varying between 5.5-6.5 were obtained. Ionization of a modulatory group with pKa 5.5 alone modifies the reaction mechanism of AcChE, and binding of substrates and inhibitors at this site provides modulation of catalysis/binding at the active center. Analysis of the relative magnitudes of pH-independent rate constants for the two reactive states revealed that in terms of the overall catalysis, the EH state shows favorable reactivity towards the cationic reagents with reactivity 1.0, as compared to the EH2 state with reactivities 0.25-0.55. Neutral reagents, in general, make use of the EH2 state more than cationic reagents, with reactivities 1.0 for the EH state and 0.3-1.0 for the EH2 state. Further analysis showed that this discrimination between the two reactive states, by both types of reagents, occurs predominantly through the difference in binding constants K'm and Km. Relative binding of a given cationic reagent to the respective reactive states ranges from K'm = 1.8 X Km to 4.0 X Km, and from K'm = 1.0 X Km to 2.0 X Km for the neutral reagents.

Acetylcholinesterase↗
PubMed1991

General occurrence of binding to acetylcholinesterase-substrate complex in noncompetitive inhibition and in inhibition by substrate.

To assess the relative importance of binding to enzyme-substrate complex (E.S) and to acetylenzyme (EA), noncompetitive inhibition has been studied in hydrolysis by acetylcholinesterase (AcChE) of cationic and uncharged substrates - acetylcholine (AcCh), 3,3-dimethylbutyl acetate, n-butyl acetate, 2-(methylammonio)ethyl acetate, 2- (N,N-diethyl-N-n-butylammonio)ethyl acetate (DEBAAc) and 2-(methylsulfonyl)ethyl acetate. For the N-trimethyl quaternary ions related to AcCh, tetramethylammonium ion, choline and choline ethyl ether, noncompetitive inhibition (Ki(nonc) is more favorable with the slower substrates than with AcCh, i.e., when E.S greater than EA, and is attributed to formation of enzyme-substrate-inhibitor complexes, E.S.I'. Noncompetitive inhibition by tetraethyl-, tert-butyl- and isopropylammonium ions, and acetamidocholine and its lower dimethyl analogue, is also attributed to E.S.I' complexes. Peripheral binding of these inhibitors decreases acylation more than deacylation. Some tertiary dimethylamonio ions have more favorable Ki(nonc) values with AcCh, decreasing deacylation more than acylation. The substrate DEBAAc is a more effective noncompetitive than competitive inhibitor in hydrolysis of AcCh, indicating that it binds more strongly in a peripheral site than in the active site of the free enzyme. In its hydrolysis by AcChE, it acts as its own noncompetitive inhibitor, by this non-productive binding. Formation of E.S.I' complexes is a general characteristic of hydrolysis by AcChE and decrease in rates at high concentrations of AcCh and related substrates is attributed to peripheral regulatory site binding, formation of E.S.S' complexes, rather than to binding to the acetylenzyme.

Acetates↗
PubMed1989

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate.

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.

Acetylcholine↗
PubMed1988

Supracrystallographic resolution of interactions contributing to enzyme catalysis by use of natural structural variants and reactivity-probe kinetics.

1. The influence on the reactivities of the catalytic sites of papain (EC 3.4.22.2) and actinidin (3.4.22.14) of providing for interactions involving the S1-S2 intersubsite regions of the enzymes was evaluated by using as a series of thiol-specific two-hydronic-state reactivity probes: n-propyl 2-pyridyl disulphide (I) (a 'featureless' probe), 2-(acetamido)ethyl 2'-pyridyl disulphide (II) (containing a P1-P2 amide bond), 2-(acetoxy)ethyl 2'-pyridyl disulphide (III) [the ester analogue of probe (II)] and 2-carboxyethyl 2'-pyridyl disulphide N-methylamide (IV) [the retroamide analogue of probe (II)]. Syntheses of compounds (I), (III) and (IV) are reported. 2. The reactivities of the two enzymes towards the four reactivity probes (I)-(IV) and also that of papain towards 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide (VII) (containing both a P1-P2 amide bond and an L-phenylalanyl side chain as an occupant for the S2 subsite), in up to four hydronic (previously called protonic) states, were evaluated by analysis of pH-dependent stopped-flow kinetic data (for the release of pyridine-2-thione) by using an eight-parameter rate equation [described in the Appendix: Brocklehurst & Brocklehurst (1988) Biochem. J. 256, 556-558] to provide pH-independent rate constants and macroscopic pKa values. The analysis reveals the various ways in which the two enzymes respond very differently to the binding of ligands in the S1-S2 intersubsite regions despite the virtually superimposable crystal structures in these regions of the molecules. 3. Particularly striking differences between the behaviour of papain and that of actinidin are that (a) only papain responds to the presence of a P1-P2 amide bond in the probe such that a rate maximum at pH 6-7 is produced in the pH-k profile in place of the rate minimum, (b) only in the papain reactions does the pKa value of the alkaline limb of the pH-k profile change from 9.5 to approx. 8.2 [the value characteristic of a pH-(kcat./Km) profile] when the probe contains a P1-P2 amide bond, (c) only papain reactivity is affected by two positively co-operative hydronic dissociations with pKI congruent to pKII congruent to 4 and (d) modulation of the reactivity of the common -S(-)-ImH+ catalytic-site ion-pair (Cys-25/His-159 in papain and Cys-25/His-162 in actinidin) by hydronic dissociation with pKa approx. 5 is more marked and occurs more generally in reactions of actinidin than is the case for papain reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Catalysis↗
PubMed1987

Differences in the chemical and catalytic characteristics of two crystallographically 'identical' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)

Acylation↗
PubMed1987

Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding.

1. 2-(N'-Acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [compound (III)] and 2-(acetamido)ethyl 2'-pyridyl disulphide [compound (IV)] were synthesized by acylation of the common intermediate, 2-aminoethyl 2'-pyridyl disulphide, to provide examples of chromogenic thiol-specific substrate-derived two-protonic-state electrophilic probe reagents. These two reagents, together with n-propyl 2-pyridyl disulphide [compound (II)], provide structural variation in the non-pyridyl part of the molecule from a simple hydrocarbon side chain in compound (II) to a P1-P2 amide bond in compound (IV) and further to both a P1-P2 amide bond and a hydrophobic side chain (of phenylalanine) at P2 as a potential occupant of S2 subsites. 2. These disulphides were used as reactivity probes to investigate specificity and binding-site-catalytic-site signalling in a number of cysteine proteinases by determining (a) the reactivity at pH 6.0 at 25 degrees C at I 0.1 of compound (III) (a close analogue of a good papain substrate) towards 2-mercaptoethanol, benzimidazol-2-ylmethanethiol [compound (V), as a minimal catalytic-site model], chymopapains B1-B3, chymopapain A, papaya proteinase omega, actinidin, cathepsin B and papain, (b) the effect of changing the structure of the probe as indicated above on the reactivities of compound (V) and of the last five of these enzymes, and (c) the forms of pH-dependence of the reactivities of papain and actinidin towards compound (III). 3. The kinetic data suggest that reagents of the type investigated may be sensitive probes of molecular recognition features in this family of enzymes and are capable not only of detecting differences in binding ability of the various enzymes but also of identifying enzyme-ligand contacts that provide for binding-site-catalytic-site signalling mechanisms. 4. The particular value of this class of probe appears to derive from the possibility of activating the 2-mercaptopyridine leaving group not only by formal protonation, as was recognized previously [see Brocklehurst (1982) Methods Enzymol. 87C, 427-469], but also by hydrogen-bonding to the pyridyl nitrogen atom when the appropriate geometry in the catalytic site is provided by enzyme-ligand contacts involving the non-pyridyl part of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

2,2'-Dipyridyl↗
PubMed1986

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.

2,2'-Dipyridyl↗
PubMed1984

Comparative resonance Raman spectroscopic and kinetic studies of acyl-enzymes involving papain, actinidin and papaya peptidase II.

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.14) and papaya peptidase II (the more basic monothiol cysteine proteinase of Carica papaya). The acyl groups are N-benzoylglycine and N-(beta-phenylpropionyl)glycine containing C = S or 13C = S at the ester function. Comparison of the data with those for the corresponding papain (EC 3.4.22.2) analogues [Storer, Lee & Carey (1983) Biochemistry 22, 4789-4796] allows us to define the conformation of the dithioacyl group in the catalytic site. In each case the dithioacyl group is bound in a single conformation known as conformer B, in which the glycinic nitrogen atom comes into close contact with the sulphur atom of the catalytic-site cysteine residue. For the N-(beta-phenylpropionyl)glycine dithioacyl-enzymes the torsional angles of the NH-CH2-C(= S) bonds assume values typical of an essentially relaxed non-strained state. However, for the N-benzoylglycine dithioacyl-enzymes there is evidence for a slightly perturbed conformer B, and the perturbation is most pronounced for N-benzoylglycine dithioacyl-actinidin. Values of k+2/Ks and k+3 for the reactions of papain, actinidin and papaya peptidase II with N-benzoylglycine and N-(beta-phenylpropionyl)glycine methyl thionoesters were obtained by a pre-steady-state kinetic study. Wide variation was found in k+2/Ks, but the values of k+3 are all similar. This general picture is supported by the results from a steady-state kinetic study of the reactions of the three enzymes with N-benzoyl-L-arginine-p-nitroanilide and with N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester. The similarity of the values of k+3, together with the invariance of conformer B geometry at the P1 site, suggests that the chemistry of the deacylation process is highly conserved among these three cysteine proteinases.

Acylation↗
PubMed1984

Differences between the electric fields of the catalytic sites of papain and actinidin detected by using the thiol-located nitrobenzofurazan label as a spectroscopic reporter group.

The catalytic-site thiol groups of papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were each labelled with the nitrobenzofurazan (Nbf) chromophore by reaction with 4-chloro-7-nitrobenzofurazan at pH 4.4. The electronic-absorption spectra of both labelled enzymes were determined in aqueous solution, in the pH ranges approx. 2-5 for S-Nbf-papain and approx. 3.3-8 for S-Nbf-actinidin, and for the latter also in 6 M-guanidinium chloride. The spectrum of S-Nbf-papain is characterized by lambda max. = 402 nm at pH 5 and by lambda max. = 422 nm at pH 2.18. The pH-dependent shift in lambda max. accompanies a pH-dependent change in A 430, the nature of which is consistent with its dependence on a single ionizing group with pKa 3.7. The spectrum of S-Nbf-actinidin is pH-independent in the pH range approx. 3.3-8 and is characterized by lambda max. = 413 nm. This absorption maximum shifts to 425 nm in 6M-guanidinium chloride. These results are discussed and related to those reported previously from studies on papain and actinidin with various reactivity probes. Despite the close similarity in the catalytic sites of papain and actinidin deduced from X-ray-diffraction studies, the considerable differences in their reactivity characteristics are mirrored by differences in their electric fields detected by the Nbf spectroscopic label. The microenvironment in the catalytic site of actinidin appears to favour the existence of ions significantly more than in the corresponding region in papain.

4-Chloro-7-nitrobenzofurazan↗